Stem cells play central roles in tissue homeostasis, wound repair, and regeneration, but also represent an important cell type of origin for diseases such as cancer. The mouse prostate is an excellent system for investigating the function and molecular regulation of stem/progenitor cells in the contexts of tissue organogenesis, homeostasis, and regeneration. In this competing renewal application, we will investigate the roles of prostate epithelial stem cells in these functional contexts using in vivo methods. In the current funding period, we have used genetic lineage-tracing to identify a prostate epithelial stem cell population, termed castration-resistant Nkx3.1 expressing cells (CARNs), which displays multipotency and self-renewal and is a cell of origin for prostate cancer. In our preliminary studies, we have now shown that CARNs are predominantly localized to ductal tips, consistent with their stem cell properties during prostate regeneration, and that Nkx3.1 high-expressing cells (NHCs) are similarly localized to ductal tips during organogenesis, suggesting that NHCs correspond to tissue-forming prostate progenitors. Furthermore, we have used molecular profiling approaches to identify novel markers of CARNs, and have initiated genetic lineage-tracing studies to investigate the stem cell properties of adult prostate epithelial basal cells in vivo. Based on our preliminary findings, we will now pursue a comprehensive research program to understand the identity and function of stem/progenitor cells during prostate organogenesis and adulthood, using in vivo approaches. Consequently, we propose four linked specific aims: (1) Functional analysis of NHCs in organogenesis and adult tissue homeostasis to determine whether cells that express high levels of Nkx3.1 have stem/progenitor properties in the neonatal as well as adult prostate; (2) Functional analysis of CARNs in regeneration to investigate the epithelial lineage hierarchy, examine the relationship between neonatal and adult CARNs, and perform quantitative studies of the clonal fate of CARN progeny; (3) Molecular analysis of prostate epithelial progenitors to identify markers of CARNs and NHCs as well as signaling pathways that modulate their functional properties; and (4) Investigation of the progenitor potential of prostate basal epithelial cells to determine whether basal cells display stem cell properties similar to or different from CARNs/NHCs during organogenesis, regeneration, and/or tissue homeostasis. Taken together, these studies will provide key insights into prostate epithelial stem cell function in vivo, and will have important implications for mammalian development, stem cell biology, and human cancer. PUBLIC HEALTH RELEVANCE: Our studies should have significant translational implications, since the elucidation of prostate stem/progenitor identity and function as well as the epithelial lineage hierarchy is of prime importance for understanding the cell of origin for human prostate disease and cancer. In particular, differences in the cell of origin within the lineage hierarchy may represent the basis for distinct tumor subtypes that differ in treatment response and outcome. Moreover, understanding normal stem cell properties is likely to be crucial in developing targeted therapeutics for putative cancer stem cells.